Chlorohydrin and hydroxycarboxylic ester asymmetric hydrolase gene

ABSTRACT

The present invention is to provide a gene having asymmetric hydrolase activity which is useful for synthesis of an optically active carboxylic acid, its antipode ester, and lactone, and a hydroxycarboxylic ester asymmetric hydrolase enzyme (EnHCH) derived from  Enterobacter sp . DS-S-75 strain (FERM BP-5494) which is bacteria belonging to the genus  Enterobacter , a EnHCH gene shown by base sequence of SEQ.ID.NO: 1, a gene encoding a protein having an amino acid sequence of SEQ.ID.NO: 2, and  E. coli  DH5α (pKK-EnHCH) deposited to International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology as a deposition No. FERM BP-08466.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a hydroxy carboxylic ester asymmetrichydrolase (Hydroxy Carboxylic ester Hydrolase: hereinafter abbreviatedto as “EnHCH”), a gene encoding the same, a recombinant vectorcontaining the gene, a transformant transformed by the recombinantvector and a process for producing the enzyme using the transformant,which EnHCH is a biological catalyst useful for the preparation of anoptically active chlorohydrin, optically active3-hydroxy-γ-butyrolactone, optically active hydroxycarboxylic acid, andits antipode alkyl ester which is a useful chiral building block in thesynthesis of an optically active compound to be used for medicines,agricultural chemicals, and strongly dielectic liquid crystal, etc.Moreover, it relates to a process for the preparation of an opticallyactive material using the enzyme and the transformant.

2. Prior Art

Optically active compounds have been usually produced by a chemicalsynthesis comprising converting the corresponding optically activestarting compound into the desired compound, or by an optical resolutioncomprising treating the corresponding racemic compound with an opticallyresolving agent, but recently, it is reported to produce the opticallyactive compound by a biological optical resolution utilizing anasymmetric reduction or asymmetric hydrolysis of a racemic compound witha microorganism or an enzyme.

As a method of preparing optically active 4-chloro-3-hydroxycarboxylicester, E. Santaniello, et al. have reported a process for prepaingS-ethyl 4-chloro-3-hydroxybutanoate from ethyl 4-chloro-3-oxobutanoateby asymmetric reduction using baker's yeast (E. Santaniello et al.,Journal of Chemical Research (J. Chem. Research), 1984, pp. 132–133).Also, Takahashi et al. have reported a process for the preparation ofoptically active ethyl 4-chloro-3-hydroxybutanoate from ethyl4-chloro-3-oxobutanoate by asymmetric reduction using microorganisms(Japanese Laid-Open Patent Application No. Sho. 61–146191).

As a preparation process using an enzyme, Peters et al. have reported aprocess for the preparation of S-methyl 3-hydroxybutanoate and R-ethyl4-chloro-3-hydroxybutanoate from methyl 3-oxobutanoate or ethyl4-chloro-3-oxobutanoate by asymmetric reduction using carbonyl reductasefor Rhodococcus erythropolis (J. Peters et al., Applied MicrobiologyBiotechnology (Appl. Microbiol. Biotechnol.), 1992, vol. 38, pp.334–340, T. Zelinski et al., Journal of Biotechnology (J. Biotechnol.),1994, vol. 33, pp. 283–292). Moreover, Shimizu et al. have reported apreparation process of R-ethyl 4-chloro-3-hydroxybutanoate by asymmetricreduction using aldehyde reductase for Sporoboromyces salmonicolorAKU4429 strain (Shimizu et al., Biotechnology Letter (Biotechnol.Lett.), 1990, vol. 12, pp. 593–596, Shimizu et al., Applied MicrobiologyBiotechnology (Appl. Microbiol. Biotechnol.), 1990, vol. 56, pp.2374–2377).

However, in the preparation method of an optically active β-hydroxyester compound from a prochiral β-keto ester compound by asymmetricreduction using these microorganisms or enzymes, an expensive coenzymesuch as NADH (nicotinamide adenine dinucleotide) or NADPH (nicotinamideadenine dinucleotide phosphate), etc. are required for the reaction, andits oxidized product is required to be converted again into a reducedmaterial whereby an enzyme such as glucose oxidase or formic aciddehydrogenase, etc. is separately required. Moreover, the above reactionstep becomes a rate-determining reaction, and thus, the above-mentionedprocess cannot be said to be an industrially useful process.

As a process for the preparation of optically active 3-hydroxybutanoate,it has been known a process for the preparation of S-3-hydroxybutanoatefrom acetoacetate by asymmetric reduction using microorganisms such asyeast (Hamdani et al., Tetrahedron: Asymmetery, 1991, vol. 2, pp.867–870) or Halobacterium halobium, (Ehrler and Seebach, HelveticaChimica Acta (Helv. Chim. Acta), 1989, vol. 72, pp. 793–799). However,in this method, an expensive coenzyme such as NADH (nicotinamide adeninedinucleotide) or NADPH (nicotinamide adenine dinucleotide phosphate),etc. are required for the reaction.

When the above-mentioned Halobacterium halobium is acted on racemic3-hydroxybutanoate, it has been reported that R-3-hydroxybutanoateremains by carboxylate hydrolysis reaction. It has been not known,however, a process for the preparation of S-3-hydroxybutanoate with highoptical purity from racemic 3-hydroxybutanoate using a microorganismhaving a stereospecific ester resolution activity.

As a process for the preparation of optically active 2-hydroxybutanoate,it has been known a process from 2-hydroxyhexadecanoic ester or2-hydroxytetracosanoic ester by an interesterification reaction of asecondary alcohol using lipase (Sugai et al., Yukigosei Kagakukaishi(Journal of Organic Syntesis Chemistry Association), 1995, vol. 53, pp.48–58). However, it has been not known a process for the preparation ofoptically active 2-hydroxybutanoate by hydrolysis reaction of acarboxylate.

As a process for the preparation of optically active lactic acidutilizing microorganisms or enzymes, there have been known afermentation method from glucose using lactic acid bacterium (Brin,Biochemical Preparation (Biochem. Prepn.), 1953, vol. 3, p. 61; Andersenand Greaves, Industrial Engineering Chemistry (Ind. Eng. Chem.), 1942,vol. 34, p. 34) or a preparation process from 2-halopropionic acid by adehalogenation enzyme using a Pseudomonas genus microorganism (Soda etal., Biodegradation, 1995, vol. 6, pp. 223–227). However, it has beennot known a process for the preparation of optically active lactate orlactic acid by stereospecifically hydrolyzing a carboxylate.

As a process for the preparation of optically activetetrahydrofuran-2-carboxylate by utilizing an enzyme, it has been knowna process for the preparation of the same from a racemic mixture bystereospecific hydrolysis using various kinds of protease, lipase (WO01/92553-A, WO 01/92554-A), esterase (Japanese Laid-Open PatentApplication No. 2002-171994) each derived from nature. However, there isno process which can obtain R-methyl tetrahydrofuran-2-carboxylate withhigh optical purity. Also, the above process requires an expensiveenzyme.

By the reasons as stated above, it has been strongly desired to culturea separated strain, to produce an enzyme with high properties with aninexpensive cost and a large amount whereby applying the enzyme to aprocess for the preparation of optically active hydroxycarboxylic acidand its antipode alkyl ester to make the process simple and ease.

Also, if a gene of the enzyme can be subjected to cloning, it ispossible to produce the enzyme with an inexpensive cost and a largeamount by using a genetic engineering technique, so that is has beenstrongly desired to subject the gene encoding the enzyme to cloning.

As a process for solving the above problems, the present inventors havealready found Enterobacter sp. DS-S-75 strain (FERM BP-5494) separatedfrom soil which is a bacterium belonging to the genus Enterobacter andhaving an activity of subjecting to stereoselective ester hydrolysisreaction and proposed a process for convertingS-4-chloro-3-hydroxybutanoate into optically active3-hydroxy-γ-butyrolactone by acting microorganisms or a product thereofon a chlorohydrin compound such as racemic 4-chloro-3-hydroxybutanoateto effect stereoselective dechlorination and ester hydrolysis reaction,and to recover a remaining another R-4-chloro-3-hydroxybutanoatesimultaneously (see Japanese Laid-Open Patent Application No. Hei.9-47296 and U.S. Pat. No. 5,776,766, Suzuki et al., Enzyme and MicrobialTechnology, 19.99, vol. 24, pp. 13–20).

They have further studied about a substrate specificity in theabove-mentioned microorganism raction, and as a result, they have foundthat the microorganisms have characteristics of stereoselectivelydegrading various kinds of hydroxycarboxylic esters (Japanese PatentApplication No. Hei. 13-391726). They have further studied, and as aresult, they have succeeded in purifying asymmetric hydrolase (EnHCH)which participates in the above-mentioned microorganism reaction fromthe cells of the microorganism and in obtaining a EnHCH gene encodingthe same.

SUMMARY OF THE INVENTION

The present invention is to provide a chlorohydrin and hydroxycarboxylicester asymmetric hydrolase (EnHCH) and a gene thereof in whichstereoselectivity is improved, which can markedly improve catalyticactivity of a microorganism as compared with the conventional one, andcan produce an optically active compound with an industrial scale byusing microorganisms prepared by genetic engineering. The presentinvention is also to provide a vector including the gene, atransformant, and a process for producing an optically active isomerusing the enzyme. The present invention is further to provide a genewhich encodes a signal peptide of EnHCH which stably express gene in thetransformant.

According to the present invention, it is provided a gene comprisingeither one of base sequences selected from:

(a) a base sequence described in SEQ.ID.NO: 1,

(b) a base sequence which encodes a protein having a chlorohydrin andhydroxycarboxylic ester asymmetric hydrolase activity where the basesequence is a base sequence in which one or a plural number of base(s)is/are deleted, added or substituted from the base sequence described inSEQ.ID.NO: 1,

(c) a base sequence which encodes a protein having a chlorohydrin andhydroxycarboxylic ester asymmetric hydrolase activity and hybridizeswith the base sequence described in SEQ.ID.NO: 1 under stringentconditions,

(d) a base sequence which encodes an amino acid sequence described inSEQ.ID.NO: 2,

(e) a base sequence which encodes an amino acid sequence having achlorohydrin and hydroxycarboxylic ester asymmetric hydrolase activitywhere the amino acid sequence is an amino acid sequence in which one ora plural number of amino acid(s) is/are deleted, added or substitutedfrom the amino acid sequence described in SEQ.ID.NO: 2, and

(f) a base sequence which encodes an amino acid sequence having achlorohydrin and hydroxycarboxylic ester asymmetric hydrolase activitywhere the amino acid sequence is an amino acid sequence having 60% ormore of homology with the amino acid sequence described in SEQ.ID.NO: 2.

Also, according to the present invention, it is provided a proteincomprising either one of amino acid sequence selected from:

(a) an amino acid sequence described in SEQ.ID.NO: 2,

(b) an amino acid sequence having a chlorohydrin and hydroxycarboxylicester asymmetric hydrolase activity where the amino acid sequence is anamino acid sequence in which one or a plural number of amino acid(s)is/are deleted, added or substituted from the amino acid sequencedescribed in SEQ.ID.NO: 2, and

(c) an amino acid sequence having a chlorohydrin and hydroxycarboxylicester asymmetric hydrolase activity where the amino acid sequence is anamino acid sequence having 60% or more of homology with the amino acidsequence described in SEQ.ID.NO: 2.

According to the present invention, it is provided a signal peptidecomprising an amino acid sequence described in SEQ.ID.NO: 7 for aresting strain reaction using a transformant of the present invention.

According to the present invention, it is provided a DNA which encodes asignal peptide comprising a base sequence described in SEQ.ID.NO: 8.

According to the present invention, it is provided a vector containingthe gene of the present invention. The vector preferably contains theDNA which encodes a signal peptide, and is further preferably plasmidpKK-EnHCH1.

According to the present invention, it is provided a transformantcontaining the above-mentioned vector. The host is preferably E. coli,more preferably E. coli JM109 strain or DH5α strain.

According to the present invention, it is provided a process for thepreparation of the protein of the present invention which comprisesusing the gene or protein of the present invention.

According to the present invention, it is further provided a process forthe preparation of an optically active compound using the protein ortransformant of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing showing EnHCH gene (pBl-EnHCH) which hasbeen subjected to cloning to pBluescriptIIKS obtained by colonyhybridization.

FIG. 2 is an explanation of a PCR primer for construction of pKK-EnHCH 1to 4.

FIG. 3 shows subcloning of a PCR product obtained in FIG. 2 to a plasmidpKK223-3.

FIG. 4 is a schematic drawing of a SDS-PAGE photograph of a recombinantprotein.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following, embodiments of the present invention are explained inmore detail.

The present invention relates to a gene comprising either one of basesequences selected from:

(a) a base sequence described in SEQ.ID.NO: 1,

(b) a base sequence which encodes a protein having a chlorohydrin andhydroxycarboxylic ester asymmetric hydrolase activity where the basesequence is a base sequence in which one or a plural number of base(s)is/are deleted, added or substituted from the base sequence described inSEQ.ID.NO: 1,

(c) a base sequence which encodes a protein having a chlorohydrin andhydroxycarboxylic ester asymmetric hydrolase activity and hybridizeswith the base sequence described in SEQ.ID.NO: 1 under stringentconditions,

(d) a base sequence which encodes an amino acid sequence described inSEQ.ID.NO: 2,

(e) a base sequence which encodes an amino acid sequence having achlorohydrin and hydroxycarboxylic ester asymmetric hydrolase activitywhere the amino acid sequence is an amino acid sequence in which one ora plural number of amino acid(s) is/are deleted, added or substitutedfrom the amino acid sequence described in SEQ.ID.NO: 2, and

(f) a base sequence which encodes an amino acid sequence having achlorohydrin and hydroxycarboxylic ester asymmetric hydrolase activitywhere the amino acid sequence is an amino acid sequence having 60% ormore of homology with the amino acid sequence described in SEQ.ID.NO: 2.

In the present specification, the terms “one or a plural number of aminoacid(s) is/are deleted, added or substituted” mean that, for example, anoptional number of 1 to 20 amino acids, preferably 1 to 15 amino acids,more preferably 1 to 10 amino acids, further preferably 1 to 5 aminoacids is/are deleted, added or substituted. In the presentspecification, the terms “one or a plural number of base(s) is/aredeleted, added or substituted” mean that, for example, an optionalnumber of 1 to 20 bases, preferably 1 to 15 bases, more preferably 1 to10 bases, further preferably 1 to 5 bases is/are deleted, added orsubstituted.

In the present specification, the terms “which can hybridize understringent conditions” mean a nucleic acid obtained by using a colonyhybridization method, a plaque hybridization method, or a southern blothybridization method, etc. which use a nucleic acid such as DNA or RNAas a probe. More specifically, there may be mentioned a DNA which isidentifiable by subjecting to hybridization using a DNA derived from acolony or plaque, or a filter to which a fragment of the DNA is fixed,in the presence of 0.7 to 1.0M NaCl at 65° C., and then, washing thefilter with 0.1 to 2-fold of a SSC solution (a composition of the SSCsolution with a 1-fold concentration comprises 150 mM of sodium chlorideand 15 mM of sodium citrate) at 65° C. The hybridization can be carriedout according to the method as described in Molecular Cloning: ALaboratory Mannual, 2nd Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989. (hereinafter abbreviated to as “Molecular Cloning2nd Ed.”) and the like.

As the DNA which can hybridize under stringent conditions, there may bementioned a DNA having homology with a certain degree or more with abase sequence of the DNA to be used as a probe, and the homology is, forexample, 60% or more, preferably 70% or more, more preferably 80% ormore, further preferably 90% or more, particularly preferably 95% ormore, most preferably 98% or more.

In the present specification, the terms “chlorohydrin andhydroxycarboxylic ester asymmetric hydrolase” mean a protein havingamino acid sequence of SEQ.ID.NO: 2 with a molecular weight of 75.0 kDa,which is a homodimer having a molecular weight of 37.5 kDa, with anisoelectric point of 6.7.

In the present specification, the terms “chlorohydrin andhydroxycarboxylic ester asymmetric hydrolase activity” mean an activitywhich is to subject to stereoselectively dechlorination and hydrolysisof a racemic chlorohydrin represented by the following formula [1]:Cl—CH₂—CHOH—CH₂—COOR¹  [1]

-   -   wherein R¹ represents a C₁ to C₄ alkyl group, to form        S-3-hydroxy-γ-butyrolactone represented by the formula [2]:

and to remain R-chlorohydrin; to subject to hydrolysis stereoselectivelyof a racemic hydroxycarboxylic ester represented by the followingformula [3]:R²—(CH₂)_(n)—CHOH—(CH₂)_(m)—COOR¹  [3]

-   -   wherein R¹ represents a C₁ to C₄ alkyl group; R² represents a        methyl group or a benzyl group; when R² is a methyl group, m is        0 and n is 0 or 1, or m is 1 and n is 0; and when R² is a benzyl        group, m is 0 and n is 1;        to form an optically active hydroxycarboxylic acid and to remain        its antipode ester; or to subject to hydrolysis        stereoselectively of a racemic tetrahydrofuran-2-carboxylic        ester represented by the following formula [4]:

-   -   wherein R¹ represents a C₁ to C₄ alkyl group, to remain        R-tetrahydrofuran-2-carboxylic ester.

A method of obtaining a gene of the present invention is notspecifically limited. An objective gene can be isolated by preparing asuitable probe or primer based on information of the base sequence ofSEQ.ID.NO: 1 or the amino acid sequence of SEQ.ID.NO: 2 disclosed in thepresent specification, and subjecting to screening of a DNA library inwhich said gene is expected to be present by using the above probe orprimer.

More specifically, the EnHCH gene shown in SEQ.ID.NO: 1 of the presentinvention can be separated from a chromosome DNA of a microorganismDS-S-75 strain (FERM BP-5494) belonging to the genus Enterobacter. TheDNA fragment having the EnHCH gene can be obtained from the gene donor,for example, based on the partial amino acid sequence of a polypeptidechain of the purified enzyme EnHCH. That is, the purified enzyme isdigested by endopeptidase, a part of the amino acid sequence of therespective fagments is determined by a protein sequencer, and a primerfor the polymerase chain reaction (PCR) is synthesized based thereon.Then, PCR is carried out using a chromosome DNA of a DS-S-75 strainbelonging to the genus Enterobacter as a template to amplify a part ofthe EnHCH gene whereby the base sequence is clarified. By using theobtained partial base sequence of the EnHCH gene as a probe, a DNAfragment of the objective gene can be obtained by using thehybridization method from the DNA library prepared from the chromosomeDNA of the gene donor according to the conventional manner.

As a method for determining the DNA base sequence of the obtained EnHCHgene, a dideoxy sequence method may be mentioned. This method includesvarious kinds of methods generally used in the field of geneticengineering such as a method of amplifying a gene by PCR or a method ofdeleting a nucleic acid(s) by a nuclease, or the like. According tothese methods, an open reading flame (ORF) which encodes whole aminoacids in the objective DNA base sequence shown by SEQ.ID.NO: 1 can beconfirmed.

The DNA which encodes EnHCH of the present invention is characterized inthat an amino acid sequence having an EnHCH activity substantiallycontains a base sequence which encodes the polypeptide shown inSEQ.ID.NO: 2. Here, as long as it has an EnHCH activity, one or severalnumber of amino acid(s) may be deleted, added or substituted in theamino acid sequence shown in SEQ.ID.NO: 2. For example, modification ofDNA can be optionally carried out by the method known in the art such asa site-specific mutation introducing method using a syntheticoligonucleotide, so that some of the amino acids constituting the aminoacid sequence which encodes the DNA are deleted, added or substituted.Also, by using the DNA shown in SEQ.ID.NO: 1 or a DNA which is preparedby optionally modifying the above DNA as a template, a DNA in whichmutation is introduced at random can be obtained by effecting a PCRmethod in the presence of Mn²⁺ ion (generally a concentration of 0.5 to10 mM), or making a concentration of a specific nucleotide low. Of theseDNAs thus obtained, it is needless to say that a material which encodesa protein having an EnHCH activity is included in the present invention.

Also, the present invention relates to a protein comprising either oneof amino acid sequence selected from:

(a) an amino acid sequence described in SEQ.ID.NO: 2,

(b) an amino acid sequence having a chlorohydrin and hydroxycarboxylicester asymmetric hydrolase activity where the amino acid sequence is anamino acid sequence in which one or a plural number of amino acid(s)is/are deleted, added or substituted from the amino acid sequencedescribed in SEQ.ID.NO: 2, and

(c) an amino acid sequence having a chlorohydrin and hydroxycarboxylicester asymmetric hydrolase activity where the amino acid sequence is anamino acid sequence having 60% or more of homology with the amino acidsequence described in SEQ.ID.NO: 2.

In the present specification, “an amino acid sequence having 60% or moreof homology with the amino acid sequence described in SEQ.ID.NO: 2” isnot specifically limited so long as it has homology with the amino acidsequence of SEQ.ID.NO: 2 of 60% or more, for example, it means 60% ormore, preferably 70% or more, more preferably 80% or more, furtherpreferably 90% or more, particularly preferably 95% or more, mostpreferably 98% or more.

A purification method of the enzyme according to the present inventionis not specifically limited, and purification can be carried out byoptionally combining purification methods generally employed in the art.For example, after crushing cells of the microorganisms by ultrasonicwave, salt out by ammonium sulfate is carried out, and a dissolvedmaterial of the precipitate is purified by optionally combininghydrophobic chromatography, anion exchange chromatography and gelfiltration chromatography, until it becomes single by sodiumdodecyl-sulfate-polyacrylamide gel electrophoresis (hereinafterabbreviated to as “SDS-PAGE”).

The present invention also relates to a signal peptide comprising 25amino acids shown in SEQ.ID.NO: 7 which is located at upstream of theEnHCH and a gene encoding the same (shown by SEQ.ID.NO: 8). According tothis signal peptide, a transformant of E. coli into which the above geneis introduced having a high EnHCH activity can be obtained in the stateof a cell culture broth. When the cell is removed, the activity is lost.It means that no enzyme is present in the medium, so that it is easy toprevent from excessive reaction or to carry out extraction of theproduct. Also, when necessity arises, only the gene of said signalsequence is connected to the other useful gene.

The present invention is further to provide an expression plasmidcontaining a base sequence of a gene which encodes an amino acidsequence showing an EnHCH activity. By incorporating a necessary portionof a DNA fragment which carries gene information of EnHCH into a vector,and it is incorporated into a host cell, a transformant can be obtained.As such a vector, preferred is a material to which a suitable selectivemarker, etc. which is capable of being self-replication in a host celland is capable of selecting a recombinant host cell alone is provided,and the gene of the present invention can be expressed in a suitablehost cell. Moreover, such a vector may be those which can be easilyprepared from a conventionally known vector by a person skilled in theart using a conventionally known technique, or may be those which arecommercially available. Particularly preferred is plasmid pKK-223-3.

The present invention is further to provide a microorganism having anability of producing an EnHCH activity, which is transformed by a DNAwhich encodes the polypeptide an amino acid sequence of which issubstantially shown in SEQ.ID.NO: 2.

As a host cell to be used, any material can be used without restrictionso long as it is transformed by the resulting recombinant vector and canexpress the gene of the present inventoin. Such a host cell may includegram negative bacteria as well as gram positive bacteria so long as itcan accomplish expression of the gene of the present invention alongwith the object of the same, and further may include either of aprocaryotic cell or eucaryotic cell, or either of cells derived fromanimals or cells derived from plants.

As a host cell which can be used specifically in the present invention,there may be mentioned, for example, microorganisms selected from thegroup consisting of E. coli (Escherichia coli), the genusesEnterobacter, Saccharomyces, Xanthomonas, Acetobacter, Pseudomonas,Gluconobacter, Azotobacter, Rhizobium, Klebsiella, Salmonella andSerratia, and preferably E. coli is used. Particularly preferred is E.coli DH5α. DH5α (pKK-EnHCH1) is deposited to International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology as a deposition number FERM BP-08466.

The transformant can be obtained with a large scale by carrying outcultivation in a suitable culture medium. The culture medium may be anyof usual media which contain a carbon source, a nitrogen source,inorganic substances, etc., necessary for growth of microorganisms. Forexample, there may be used as a carbon source, hydrocarbon such asglucose, fructose, etc., an alcohol such as glycerol, mannitol,sorbitol, propylene glycol, etc., an organic acid such as acetic acid,citric acid, malic acid, maleic acid, fumaric acid, gluconic acid and asalt thereof, or a mixture thereof. As a nitrogen source, there may bementioned an inorganic nitrogen compound such as ammonium sulfate,ammonium nitrate, ammonium phosphate, etc., and an organic nitrogencompound such as urea, peptone, casein, yeast extract, meat extract,corn steep liquor, etc., and a mixture thereof. In addition, as aninorganic salt, phosphate, magnesium salt, potassium salt, manganesesalt, iron salt, zinc salt, copper salt, etc. may be added and vitaminsmay be further added, if necessary. Also, as an enzyme derived additiveto obtain a transformant cells having a high enzyme activity,antibiotics such as ampicillin, kanamycin, chloramphenicol, etc. may beadded to a culture broth to express an objective DNA effectivelydepending on the strain to be used in a nutrient medium such as theabove-mentioned medium, peptone medium, bouillon medium, etc., or anactivation inducer of a promoter such as isopropylβ-D(−)-thiogalactopyranoside (IPTG), etc. may be used. Cultivation maybe carried out under aerobic conditions by controlling an optional rangeat a pH of 4 to 10 and a temperature of 20 to 60° C. for 1 to 5 days,and it is more effective to carry out cultivation under optimumconditions for the transformant to be used.

Extraction of the enzyme from the resulting cells of the EnHCH-producingtransformant can be carried out by the following method. According tothe methods of 1) a mechanical (physical) method by French press orultrasonic wave pulverization, 2) an enzyme treatment method such aslysozyme, 3) an autodissolution method, 4) an extraction methodutilizing an osmotic pressure, etc., the transformant is broken. Also,E. coli DH5α (pKK-EnHCH1) can be utilized as a high activity enzyme inthe state of a culture broth without pulverizing the cells.

The enzyme produced by the present process is not limited only to thosewhich are accorded with the polypeptide sequence of EnHCH in the originof the genus Enterobacter, and it may include any polypeptides obtainedby gene recombinant methods such as base sequence transformation or genetransformation, and show an EnHCH activity. That is, those in which oneor several number of amino acids in the peptide sequence is/are deleted,or one or several number of amino acids in the peptide sequence is/aresubstituted by other amino acid(s).

The thus produced transformant which produces a stereoselectivehydrolase with a large amount can be prepared with an industrial scalein an inexpensive medium, and it can be utilized as a bioreactor for thepreparation of R-4-chloro-3-hydroxycarboxylic ester,S-3-hydroxy-γ-butyrolactone, optically active hydroxycarboxylic acid,and its antipode ester, R-tetrahydrofuran-2-carboxylic ester, etc.

In particular, E. coli DH5α (pKK-EnHCH1) shows higher reactivity ascompared with a gene donor (DS-S-75 strain of the genus Enterobacter),and it is particularly possible to obtain S-3-hydroxy-γ-butyrolactone,R-3-hydroxybutyric acid, and S-2-hydroxybutyric acid within a shortperiod of time with a high optical purity.

EXAMPLES

In the following, the present invention is explained by referring toExamples in more detail. The present invention is not at all limited bythese Examples.

Example 1 Purification of EnHCH Enzyme

Measurement of an enzyme activity was carried out by using 1% (v/v)methyl 4-chloro-3-hydroxybutanoate as a substrate and reacting in 0.5 Mpotassium phosphate buffer (pH 7.15) at 30° C., and liberated chloroions were calorimetrically determined according to the method of Iwasakiet al. (Bull. Chem. Soc. Japan, 25, 226 (1952)). An enzyme amount whichliberates 1 μmol of chloro ion within one minute is defined to be 1 U.Determination of the protein was carried out by measuring an absorptionat 280 nm.

DS-S-75 strain (FERM BP-5494) belonging to the genus Enterobacter wascultured in a PYG medium (1% peptone, 1% yeast extract and 1% glycerol,pH 7.2), and cells were prepared by centrifugation.

The resulting wet cells were pulverized by an ultra high-pressure cellpulverizing device, and then, cell residue were removed bycentrifugation to obtain cell-less extract. To the extract was addedammonium sulfate, and precipitated fraction at 50% saturation wasrecovered. Moreover, it was applied to hydrophobic chromatography byButyl-toyoperl equilibrated with 10 mM tris-sulfate buffer (pH 7.8)containing 1.6 M of ammonium sulfate. A concentration of ammoniumsulfate was lowered to 0 M, and an active fraction of the enzyme wasrecovered. To the fraction was added ammonium sulfate, and precipitatesat 80% saturation were dissolved in 10 mM tris-sulfate buffer (pH 7.8),and dialyzed by the same buffer. After dialysis, the resulting materialwas applied to ion exchange column chromatography by DEAE-sepharoseequilibrated with the same buffer. Dissolution was carried out withconcentration gradient (10–200 mM) of the same buffer, and activefractions of the enzyme were recovered. These fractions were subjectedto desalting concentration by using ultrafiltratoin membrane (Macrosep10K, available from Nihon Pall Ltd.). The concentrate was applied to gelfiltration column chromatography using Sephadex-G150 equibrated with 0.1M potassium phosphate buffer, to recover active fractions of the enzyme.

The resulting asymmetric hydrolase standard product was analyzed bySDS-PAGE, and as a result, it became a single band and the molecularweight was 37.5 kDa. As results of gel filtration column chromatographyand Native-PAGE using a separation gel to which 10–20% of concentrationgradient had been applied, the molecular weight was 75 kDa. From theseresults, it cound be found that the enzyme was homo dimer of subunitshaving a molecular weight of 37.5 kDa.

Summary of the purification was shown in Table 1, specific activity ofthe purified enzyme was 7690 U/mg-protein, and was 221-fold as comparedto the crude enzyme extract.

TABLE 1 Total Vol- protein Total Specific Purified ume weight activityactivity Yeild degree (ml) (mg) (U) (U/mg) (%) (fold) Crude 90 254088300 34.8 100 1.0 extract 0–50% 200 1680 53800 32.0 61.0 0.90 ammoniumsulfate precipitate Butyl 173 142 31800 269 43.2 7.70 toyopearl 0–80%38.3 132 23000 174 26.0 5.0 ammonium sulfate precipitate DEAE- 75.3 5.9528200 4750 32.0 136 sepharose Sephadex G- 16.8 2.54 19500 7690 22.1 221150

Example 2 Analysis of Partial Amino Acid Sequence of EnHCH Enzyme

Purified EnHCH enzyme in an amount of 45 μg was treated withchymotrypsin at 37° C. for 1 hour. SDS-PAGE was carried out by using12.5% separation gel, the resulting material was transcripted to a PVDFmembrane, and N terminus and internal partial amino acid sequences weredetermined by an amino acid sequencer. The determined amino acidsequences are shown in SEQ.ID.NO: 3 and 4.

Example 3 Cloning of EnHCH Gene

(1) Preparation of Chromosome DNA from the Genus Enterobacter

DS-S-75 strain (FERM BP-5494) belonging to the genus Enterobacter wascultured in 5 ml of a PYG medium for 20 hours, and cells were recoveredby centrifugation. The cells were suspended in 490 ml of a TE solution(10 mM Tris-hydrochloride, pH 8.0, 1 mM EDTA), 30 ml 10% SDS and 50 mlof 20 mg/ml protease K were added thereto, and the mixture was reactedat 50° C. for 1 hour. Thereafter, extraction was carried out with anequal amount of phenol:chloroform:isoamyl alcohol (25:24:1), 0.1-foldamount of 3 M sodium acetate solution was added thereto, and 0.6-foldamount of 2-propanol was quietly layered. As a result, DNA generated atan interface was recovered by a glass lod by wounding it in a stringstate.

The obtained DNA was dissolved in 500 ml of a TE solution. About 1.1 mgof chromosomal DNA was obtained.

(2) Preparation of Probe

PCR primer having a DNA sequence expected from amino acid sequences ofSEQ.ID.NO: 3 and 4 determined by an amino acid sequencer was synthesized(as shown in SEQ. ID. NO: 5 and 6) by considering codon degeneration, 40cycles of PCR (thermal denaturation: 96° C. for 30 seconds, annealing:50° C. for 1 minute, elongation reaction: 72° C. for 1 minute) werecarried out by using ExTaq DNA polymerase (available from Takara ShuzoCo., Ltd.) and 50 ng of chromosome DNA extracted in (1) as a template,then, after cooling to 4° C., amplified DNA was confirmed by agarose gelelectrophoresis, and a DNA fragment was recovered by agarose gelaccording to the conventional manner. Then, the DNA fragment wassubjected to subcloning to pUC118 (available from Takara Shuzo Co.,Ltd.), and the base sequence was determined by a DNA sequencer. As aresult, in the determined DNA base sequence, a region which encodes apartial amino acid sequence (SEQ.ID.NO: 3 and 4) previously determinedcan be determined, so that the obtained amplified DNA was labeled by ³²Paccording to the conventional manner to make a probe DNA.

(3) Preparation of Restriction Enzyme Map

The chromosome DNA of the extracted DS-S-75 strain was cut by variouskinds of restriction enzymes, after subjecting to electrophoresis byusing 1% agarose gel, it was transcripted to a nitrocellulose membrane.After air drying, hybridization reaction was carried out by using theprobe prepared in (2) at 65° C. for 20 hours.

The membrane subjected to hybridization was washed with (1) 2×SSC, 65°C. for 15 minutes and (2) 1×SSC, 65° C. for 30 minutes, andautoradiogram was taken by attaching thereto an X-ray film and asensitizing paper. As a result, by comparing various signals to eachother, a restriction enzyme map at a neighbour of EnHCH gene region wasprepared.

(4) Cloning of Gene from Genome Library

Genome library in which many kinds of DNA fragments derived fromchromosome DNAs of about 6000 base pairs of DS-S-75 strains wereinserted into EcoRI portion of plasmid pBluescriptII KS (available fromTOYOBO CO., LTD.) was prepared with consideration that whole length ofthe objective EnHCH gene are contained from the restriction enzyme mapprepared in (3). 160 kinds of transformants obtained by introducing theabove into E. coli DH5α strain were replicated to a nitrocellulosemembrane, the membrane was immerced into 0.5 N NaOH to effect lysis ofbacteria, and the membrane was neutralized by immercing in 1 MTris-hydrochloride (pH 7.5). After air drying, hybridization reactionwas carried out by using the probe prepared in (2) at 65° C. for 20hours.

The membrane subjected to hybridization was washed with (1) 2×SSC, 65°C. for 15 minutes, (2) 1×SSC, 65° C. for 30 minutes and (3) 0.5×SSC, 65°C. for 1 hour, and autoradiogram was taken by attaching thereto an X-rayfilm and a sensitizing paper. As a result, one transformant whichprovides a positive signal was obtained.

(5) Determination of Base Sequence

A plasmid was extracted from the above positive strain according to theconventional manner to obtain pBl-EnHCH.

Various kinds of insertion gene fragments were deleted from thepBl-EnHCH by exonuclease III and mung bean endonuclease, base sequencesof the respective samples were determined by the dideoxy method, theobtained sequences are layerd, and base sequences were determinedbetween BamHI-PstI. The determined base sequence and the amino acidsequence estimated from the base sequence are shown in SEQ.ID.NO: 1 ofthe sequence table. A schematic drawing of pBl-EnHCH is shown in FIG. 1.In FIG. 1, Determination of the base sequence was carried out betweenBamHI-PstI portions which sandwitches ORF. ORF encodes 1086 bp 362 aminoacids. Of these, N-terminal side 25 amino acids are signal peptide.

Also, partial amino acid sequences of the purified EnHCH shown inSEQ.ID.NO: 3 and 4 existed in amino acid sequence estimated from thebase sequence of the insertion DNA fragment derived from the positivestrain, and the partial amino acid sequence was completely accored toeach other.

Example 4 Preparation of Recombinant Plasmid

ATG sequence estimated to be an initiation codon of the EnHCH gene waspresent at 3 portions. Assembly was carried out by the followingoperation, so that coupling was carried out under control of a tacpromoter of the plasmid pKK223-3 and translation was carried out fromthe above-mentioned respective initiation codon and N terminus of thepurified enzyme (FIG. 2 and FIG. 3). To provide a EcoRI recognizablesequence to just upstream of these respective ATG sequences, PCR primersshown in SEQ.ID.NO: 9 to 12 were designed, and M13 primers werecombined, 30 cycles of PCR (thermal denaturation: 96° C. for 30 seconds,annealing: 50° C. for 30 seconds, elongation reaction: 72° C. for 1minute) was carried out by using 50 ng of pBl-EnHCH1 as a template (Nterminus of the purified enzyme was substituted by methionine (FIG. 2)).In FIG. 2, with regard to three kinds of ATG sequences estimated to beinitiation codons, a PCR primer in which EcoRI was added to justupstream was designed, and PCR was carried out in combination with M13primer derived from pUC118. Also, to effect translation from valinewhich is the N terminus of the purified enzyme, PCR primer in whichvaline was substituted by methionine was designed, and PCR was carriedout in the same manner. After cooling to 4° C., amplified DNA wasconfirmed by agarose gel electrophoresis, and various kinds of DNAfragments were recovered by agarose gel according to the conventionalmanner. Then, these DNA fragments were smoothened by using a BKL kit(available from Takara Shuzo Co., Ltd.) and subjected tophosphorylation, and subjected to subcloning at a HincII restrictionenzyme portion of pUC118. With regard to the respective samples, about400 bases' sequences at upstream were determined by a DNA sequencer.

The obtained four kinds of plasmids were digested by restriction enzymesTth111I and PstI, Tth111I-PstI fragment of pBl-EnHCH was insertedaccording to the conventional manner, and further, EcoRI-PstI digestedfragment was inserted into the same portion of the pKK223-3 (availablefrom Pharmacia Co.) vector to obtain expression plasmids pKK-EnHCH1,pKK-EnHCH2, pKK-EnHCH3 and pKK-EnHCH4 (FIG. 3). In FIG. 3, subcloningwas firstly carried out to pUC118 at smooth terminus, and about 400 bpof base sequence at upstream side were determined. After confirmationthat there is no PCR elongation error, sequences between Tth111I-PstIwere replaced by those of pBl-EnHCH, and ligated by EcoRI-PstI todownstream of a tac promoter of pKK223-3.

Example 5 Preparation of Recombinant E. coli

Competent cells of E. coli JM109 and DH5α were prepared, and transformedby pKK-EnHCH to obtain recombinated E. coli JM109 (pKK-EnHCH1) and DH5α(pKK-EnHCH1). Incidentally, no DH5α (pKK-EnHCH2) could be obtained. Ascontrol samples, JM109 (pKK-223-3) and DH5α (pKK223-3) which had beentransformed only by a vector were obtained.

Example 6 Expression of EnHCH Gene in Recombinant E. coli

(1) Preparation of Culture Broth

The transformant obtained in Example 5 was cultured under aerobicconditions in a 5 ml of a LB medium (1% polypeptone, 0.5% yeast extractand 1% sodium chloride) placed in a test tube at 37° C. for 20 hours. Ascontrol samples, JM109 strain and DH5α strain each transformed bypKK223-3 were cultured in the same manner. Also, with regard to DS-S-75strain belonging to the genus Enterobacter, it was seed cultured underaerobic conditions in a 5 ml of a PYG medium (1% polypeptone, 1% yeastextract and 1% glycerin) placed in a test tube at 30° C. for 20 hours,and cultured samples were obtained from the respective strains.

(2) Evaluation of Hydrolysis Activity of Recombinant

To 250 mM Tris sulfate buffer was added p-nitrophenyl butyrate, so thatthe final concentration became 0.05% (v/v), to prepare a reactionsolution. To 3 ml of the reaction solution was added 20 ml of thecultured sample prepared as mentioned above, and the mixture was reactedat 30° C. and increase in absorbance at 400 nm derived fromp-nitrophenol which had been formed by the hydrolysis reaction wasmeasured. The results are shown in Table 2. Incidentally, 1 U representsa formed amount of 1 μmol of p-nitrophenol per 1 minute at 30° C. andspecific activity per the prepared culture broth was calculated.

TABLE 2 Specific activity Name of strain (U/ml) DS-S-75 strain belongingto 3.16 the genus Enterobacter JM109 (pKK-EnHCH1) 12.6 JM109(pKK-EnHCH2) 13.8 JM109 (pKK-EnHCH3) 2.76 JM109 (pKK-EnHCH4) 2.37 JM109(pKK223-3) 0 DH5α (pKK-EnHCH1) 34.0 DH5α (pKK223-3) 0

As a result, E. coli transformed by the pKK-EnHCH had a hydrolysisactivity, and activities of JM109 (pKK-EnHCH1), JM109 (pKK-EnHCH2) andDH5α (pKK-EnHCH1) had markedly improved as compared to that of DS-S-75strain belonging to the genus Enterobacter. In particular, hydrolysisactivity of DH5α (pKK-EnHCH1) per a culture broth was about 10.8-fold.Also, when the culture brothes of JM109 (pKK-EnHCH1) and DH5α(pKK-EnHCH1) were removed, then their activities were lost and it wasfound that no enzyme was present in the medium.

Among four strains of JM109 (pKK-EnHCH1), JM109 (pKK-EnHCH2), JM109(pKK-EnHCH3) and JM109 (pKK-EnHCH4), JM109 (pKK-EnHCH2) showed thehighest activity. Also, an SD (Shine Dalgarno) sequence to whichribosome is linked can be found at just upstream side of the second ATGsequence of the base sequence shown in FIG. 2, so that it is suggestedthat an inherent initiation codon of the EnHCH gene is to be the secondATG (SEQ.ID.NO: 1). That is, a methionine residue at the second portionof the amino acid sequence shown in FIG. 2 is the N terminus of theEnHCH to be inherently translated in the DS-S-75 strain. As a result, itis suggested that the gene comprises 1086 bp, and encodes 362 aminoacids.

(3) SDS-PAGE of Recombinant Protein

Cultured samples of JM109 (pKK-EnHCH1), JM109 (pKK-EnHCH2), JM109(pKK-EnHCH3) and JM109 (pKK-EnHCH4) prepared in (1) were harvested bycentrifugation, and after suspending with 20 mM potassium phosphatebuffer, they were pulverized by ultrasonic wave. Each 5 μg of extractedprotein samples and EnHCH purified from DS-S-75 strain belonging toEnterobacter were applied to SDS-PAGE (10% separation gel), and stainedby Coomassie Brilliant Green. As a result, in either of recombinantenzymes of the recombinant E. coli, a band of the recombinant EnHCH wasconfirmed at the same position with that of the EnHCH purified fromDS-S-75 strain belonging to Enterobacter (FIG. 4). Molecular weightmarker to be used and the molecular weight are shown below. In FIG. 4,from the lane at the left hand, the purified EnHCH derived from the genedonor (DS-S-75 strain), an extract of JM109 (pKK223-3), an extract ofJM109 (pKK-EnHCH1), an extract of JM109 (pKK-EnHCH2), an extract ofJM109 (pKK-EnHCH3), an extract of JM109 (pKK-EnHCH4), molecular weightmarkers; Phosphorylase B (97,400), bovine serum albumin (66,200),ovoalbumin (45,000), carbonic anhydrase (31,000) and tripsin inhibitor(21,500).

As shown in Example 2, N terminus amino acid of the purified enzyme wasvaline. Also, at the N terminus side of the estimated amino acidsequence of the EnHCH shown by SEQ.ID.NO: 2, many hydrophobic residues,positive-charged residues and Ala-Xaa-Ala sequence as a recognizedsequence to be cut are present. Thus, it is suggested that the aminoacid sequence from translation initiating methionine to the 25^(th)residue is to be a signal peptide (SEQ.ID.NO: 7).

(4) Analysis of N Terminus Amino Acid Sequence of Recombinant EnHCHEnzyme

A sample of cell pulverized solution of JM109 (pKK-EnHCH1) prepared in(1) and precipitated by 20% to 50% ammonium sulfate was dialyzed,subjected to SDS-PAGE using 10% separation gel, transcripted to a PVDFmembrane, and the N terminus sequence was determined by using an aminoacid sequencer. As a result of the determination of 10 residues, theywere accorded with those of the N terminus sequence of EnHCH purifiedfrom DS-S-75 strain belonging to the genus Enterobacter. Also, as aresult of SDS-PAGE of (3), bands existed at the same positions, so thatit was clarified that cleavage of the signal peptide is also done in E.coli. Also, activities of JM109 (pKK-EnHCH1) and JM109 (pKK-EnHCH2) arehigher as compared to those of JM109 (pKK-EnHCH3) and JM109(pKK-EnHCH4), so that the signal peptide can be said to be important forstable expression thereof in E. coli.

Example 7 Optical Resolution of Carboxylic Ester by Recombinant E. coli

(1) Optical Resolution of Methyl 4-chloro-3-hydroxybutanoate

In 300 ml volume of an Erlenmeyer flask was charged 60 ml of a LB mediumcontaining ampicillin, and DH5α (pKK-EnHCH1) was subjected to shakeculture at 37° C. for 16 hours. Also, as a control, DH5α (pKK223-3)transformed only by a vector was prepared. Moreover, DS-S-75 strainbelonging to the genus Enterobacter was cultured in 60 ml of a PYGmedium. To the respective culture brothes were added calcium carbonateand racemic methyl 4-chloro-3-hydroxybutanoate as substrates finalconcentrations of which became 5% (w/v) and 8% (w/v), respectively, andthe mixture was reacted at 30° C. for 1 hour under shaking. Also,DS-S-75 strain belonging to the genus Enterobacter was further reactedfor 24 hours under shaking with a substrate a final concentration ofwhich became 2%. After completion of the reaction, the cells wereremoved by centrifugation, and a concentration of the methyl4-chloro-3-hydroxybutanoate remained in the reaction mixture wasanalyzed by gas chromatography (column carrier: PEG20M, 60–80 mesh).Further, the above-mentioned cell-removed solution was extracted with anequal amount of ethyl acetate, and an optical purity of the methyl4-chloro-3-hydroxybutanoate remained in the mixture was measured by gaschromatography using G-TA (0.25 mm×30 m) manufactured by ASTEC INC. As aresult, a retension time was 14.7 minutes for R isomer and 15.7 minutesfor S isomer. Analytical conditions: column temperature: 110° C.,detector temperature: 200° C., carrier gas: nitrogen; flow rate: 0.8ml/min, detector: FID; sprit ratio: 100/1.

Moreover, the mixture was extracted twice with ethyl acetate, and theaqueous fraction was concentrated by an evaporator. The concentrate wasdehydrated by anhydrous magnesium sulfate to obtain syrup of3-hydroxy-γ-butyrolactone. To 10 ml of syrup were added 500 ml of1,2-dichloroethane and 100 ml of trifluoroacetic acid anhydride and themixture was allowed to stand for 30 minutes to carry outtrifluorination. The solvent was removed under reduced pressure, theresidue was dissolved in ethanol, and an optical purity of the3-hydroxy-γ-butyrolactone was measured by gas chromatography using G-TA(0.25 mm×30 m). A retension time was 18.7 minutes for R isomer and 19.9minutes for S isomer. Analytical conditions: column temperature: 120°C., detector temperature: 200° C., carrier gas: nitrogen; flow rate: 0.8ml/min, detector: FID; sprit ratio: 100/1.

As a result, a reaction rate of DH5α (pKK-EnHCH1) was improved ascompared to that of the gene donor (DS-S-75 strain belonging to thegenus Enterobacter). Also, it had a high stereoselectivity, and both ofR-methyl 4-chloro-3-hydroxybutanoate and formedS-3-hydroxy-γ-butyrolactone had high optical purities, and almost allamount of the S-ethyl 4-chloro-3-hydroxybutanoate had been convertedinto S-3-hydroxy-γ-butyrolactone. Also, DH5α (pKK223-3) had no opticalresolution ability. After the reaction, measured results of an opticalpurity of the remaining R-methyl 4-chloro-3-hydroxybutanoate, a yieldthereof when an amount of the racemic methyl 4-chloro-3-hydroxybutanoatebefore initiation of the reaction as 100%, and an optical purity of theformed S-3-hydroxy-γ-butyrolactone are shown in Table 3.

TABLE 3 Methyl 4-chloro-3- 3-Hydroxy-γ- hydroxybutanoate butyrolactoneOptical Optical purity purity (% ee) Yield (%) (% ee) DH5α (pKK- 99.1(R) 49.2 99.9 (S) EnHCH1) (1 hr) (substrate 8%) DH5α (pKK-223- 0 100 Notformed 3) (1 hr) (substrate 8%) DS-S-75 (1 hr) 11.8 (R) 88.4 N.D.(substrate 8%) DS-S-75 (24 hr) 99.5 (R) 48.0 95.9 (S) (substrate 8%)(2) Optical Resolution of Ethyl 3-hydroxybutanoate

Various kinds of cells were cultured in the same manner as in Example 7(1), to the respective culture brothes were added calcium carbonate andracemic ethyl 3-hydroxybutanoate as substrates final concentrations ofwhich became 5% (w/v) and 8% (w/v), respectively, and they were reactedat 30° C. for 1 hour under shaking. Also, DS-S-75 strain belonging tothe genus Enterobacter was further reacted for 4 hours under shaking.After completion of the reaction, the cells were removed bycentrifugation.

A concentration of the ethyl 3-hydroxybutanoate remained in the reactionmixture was analyzed by gas chromatography (column carrier: PEG20M,60–80 mesh). Further, the above-mentioned cell-removed solution wasextracted twice with an equal amount of ethyl acetate, and the solventof the ethyl acetate layers were removed under reduced pressure toobtain a syrup of ethyl 3-hydroxybutanoate. The obtained product wassubjected to trifluorination by trifluoroacetic anhydride, and anoptical purity thereof was measured by gas chromatography using G-TA(0.25 mm×30 m) manufactured by ASTEC INC. A retension time was 8.1minutes for R isomer and 12.1 minutes for S isomer. Analyticalconditions: column temperature: 90° C., detector temperature: 200° C.,carrier gas: nitrogen; flow rate: 0.7 ml/min, detector: FID; spritratio: 100/1.

On the other hand, a concentration of 3-hydroxybutyric acid fractionatedin the aqueous layer was also analyzed by gas chromatography (columncarrier: PEG20M, 60–80 mesh). At this time, a pH of the aqueous solutionwas made pH 4 with phosphoric acid. The aqueous layer was concentratedby an evaporator to obtain a syrup of 3-hydroxybutyric acid. In 30 mlvolume of an Erlenmeyer flask was charged 50 mg of the syrup, and underice-cooling, to the syrup were added 122 mg of 4-dimethylaminopyridine,10 ml of dichloromethane, 100 ml of ethanol and 115 mg of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Afterstirring the mixture for 10 minutes, and the mixture was further stirredat room temperature overnight under sealing. The organic layer waswashed twice with 1N hydrochloric acid and placed in a dessiccator toremove the solvent to convert the 3-hydroxybutyric acid into ethyl3-hydroxybutanoate. The resulting product was subjected totrifluorination in the same manner as mentioned above, and an opticalpurity thereof was measured by gas chromatography.

As a result, a reaction rate of DH5α (pKK-EnHCH1) was improved ascompared to that of the gene donor (DS-S-75 strain belonging to thegenus Enterobacter). Both strains had high stereoselectivity. Also, DH5α(pKK223-3) had no optical resolution ability. After the reaction,measured results of an optical purity of the remaining S-ethyl3-hydroxybutanoate, a yield thereof when an amount of the racemic ethyl3-hydroxybutanoate before initiation of the reaction as 100%, and anoptical purity of the formed R-3-hydroxybutyric acid are shown in Table4.

TABLE 4 Ethyl 3-hydroxy- 3-Hydroxy- butanoate butyric acid OpticalOptical purity purity (% ee) Yield (%) (% ee) DH5α (pKK- 99.2 (S) 49.299.9 (R) EnHCH1) (1 hr) (substrate 8%) DH5α (pKK-223- 0 100 Not formed3) (1 hr) (substrate 8%) DS-S-75 (1 hr) 37.1 (R) 72.0 N.D. (substrate8%) DS-S-75 (4 hr) 99.1 (R) 49.0 98.2 (R) (substrate 8%)(3) Optical Resolution of Ethyl 2-hydroxybutanoate by Recombinant E.coli

The reaction was carried out in the same manner as in Example 7 (2)except for changing racemic ethyl 3-hydroxybutanoate with racemic ethyl2-hydroxybutanoate. Provided that the reaction time was made 24 hoursfor the recombinant E. coli, and 24 and 36 hours for the DS-S-75 strain.After completion of the reaction, in the same manner as in Example 7(2), the concentration and the optical purity were analyzed. A retensiontime was 7.4 minutes for R isomer and 7.8 minutes for S-isomer. Opticalpurity analyses conditions: column temperature: 90° C., detectortemperature: 200° C., carrier gas: nitrogen; flow rate, 0.7 ml/min,detector: FID; sprit ratio: 100/1.

As a result, a reaction rate of DH5α (pKK-EnHCH1) was improved ascompared to that of the gene donor (DS-S-75 strain belonging to thegenus Enterobacter), and it had high stereoselectivity. After thereaction, measured results of an optical purity of the remaining R-ethyl2-hydroxybutanoate, a yield thereof when an amount of the racemic ethyl2-hydroxybutanoate before initiation of the reaction as 100%, and anoptical purity of the formed S-2-hydroxybutyric acid are shown in Table5.

TABLE 5 Ethyl 2-hydroxy- 2-Hydroxy- butanoate butyric acid OpticalOptical purity purity (% ee) Yield (%) (% ee) DH5α (pKK- 98.3 (R) 49.098.9 (S) EnHCH1) (24 hr) (substrate 8%) DH5α (pKK-223- 0 100 Not formed3) (24 hr) (substrate 8%) DS-S-75 (24 hr) 77.3 (R) 60.1 N.D. (substrate8%) DS-S-75 (36 hr) 99.2 (R) 49.3 95.1 (S) (substrate 8%)(4) Optical Resolution of Methyl Tetrahydrofuran-2-carboxylate byRecombinant E. coli

Various kinds of cells were cultured in the same manner as in Example 7(1), to the respective culture brothes were added calcium carbonate andracemic methyl tetrahydrofuran-2-carboxylate as substrates finalconcentrations of which became 5% (w/v) and 1% (w/v), respectively, andthey were reacted at 30° C. for 2 hours under shaking. Also, DS-S-75strain belonging to the genus Enterobacter was reacted in the samemanner for 4 hours and 24 hours under shaking. After completion of thereaction, the cells were removed by centrifugation.

A concentration of methyl tetrahydrofuran-2-carboxylate remained in thereaction mixture was analyzed by gas chromatography (column carrier:PEG20M, 60–80 mesh). Further, an optical purity thereof was measured bygas chromatography using G-TA (0.25 mm×30 m) manufactured by ASTEC INC.A retension time was 8.4 minutes for R isomer and 9.5 minutes forS-isomer. Analytical conditions: column temperature: 110° C., detectortemperature: 240° C., carrier gas: nitrogen; flow rate, 0.7 ml/min,detector: FID; sprit ratio: 100/1.

As a result, a reaction rate of DH5α (pKK-EnHCH1) was improved ascompared to that of the gene donor (DS-S-75 strain belonging to thegenus Enterobacter). After the reaction, measured results of an opticalpurity of the remaining R-methyl tetrahydrofuran-2-carboxylate, and ayield thereof when an amount of the racemic methyltetrahydrofuran-2-carboxylate before initiation of the reaction as 100%are shown in Table 6.

TABLE 6 Methyl tetrahydrofuran-2- carboxylate Optical purity (% ee)Yield (%) DH5α (pKK-EnHCH1) (2 hr) 98.1 (R) 39.6 (substrate 1%) DH5α(pKK-223-3) (2 hr) 0 95.0 (substrate 1%) DS-S-75 (24 hr) 95.7 (R) 54.4(substrate 1%) DS-S-75 (48 hr) 99.9 (R) 46.8 (substrate 1%)(5) Optical Resolution of Ethyl Lactate by Recombinant E. coli

Various kinds of cells were cultured in the same manner as in Example 7(1), to the respective culture brothes were added calcium carbonate andracemic ethyl lactate as substrates final concentrations of which became5% (w/v) and 6% (w/v), respectively, and they were reacted at 30° C. for24 hours under shaking. Also, DS-S-75 strain belonging to the genusEnterobacter was reacted in the same manner for 48 hours and 100 hoursunder shaking. After completion of the reaction, the cells were removedby centrifugation. A concentration of ethyl lactate remained in thereaction mixture was analyzed by gas chromatography (column carrier:PEG20M, 60–80 mesh). Further, the above-mentioned cell-removed solutionwas extracted twice with an equal amount of ethyl acetate in the samemanner as in Example 7 (1) to obtain a syrup of ethyl lactate. Theobtained product was subjected to trifluorination by trifluoroaceticanhydride, and an optical purity thereof was measured by gaschromatography using G-TA (0.25 mm×30 m) manufactured by ASTEC INC. Aretension time was 10.1 minutes for R isomer and 11.8 minutes forS-isomer. Analytical conditions: column temperature: 70° C., detectortemperature: 200° C., carrier gas: nitrogen; flow rate: 0.7 ml/min,detector: FID; sprit ratio: 100/1.

As a result, a reaction rate of DH5α (pKK-EnHCH1) was improved ascompared to that of the gene donor (DS-S-75 strain belonging to thegenus Enterobacter). After the reaction, measured results of an opticalpurity of the remaining R-ethyl lactate, and a yield thereof when anamount of the racemic ethyl lactate before initiation of the reaction as100% are shown in Table 7.

TABLE 7 Ethyl lactate Optical purity (% ee) Yield (%) DH5α (pKK-EnHCH1)(24 hr) 98.7 (R) 41.5 (substrate 6%) DH5α (pKK-223-3) (24 hr) 0 100(substrate 6%) DS-S-75 (48 hr) 33.5 (R) 65.2 (substrate 6%) DS-S-75 (100hr) 98.7 (R) 28.0 (substrate 6%)(6) Optical Resolution of Ethyl 4-phenyl-2-hydroxybutanoate byRecombinant E. coli

Various kinds of cells were cultured in the same manner as in Example 7(1), to the respective culture brothes were added calcium carbonate andracemic 4-phenyl-ethyl 2-hydroxybutanoate as substrates finalconcentrations of which became 5% (w/v) and 2% (w/v), respectively, andthey were reacted at 30° C. for 24 hours under shaking. Also, DS-S-75strain belonging to the genus Enterobacter was reacted in the samemanner for 48 hours and 100 hours under shaking. After completion of thereaction, the cells were removed by centrifugation.

A concentration of 4-phenyl-ethyl 2-hydroxybutanoate remained in thereaction mixture was analyzed by gas chromatography (column carrier:PEG20M, 60–80 mesh). Further, the above-mentioned cell-removed solutionwas extracted twice with an equal amount of ethyl acetate, and thesolvent of the ethyl acetate layer was removed under reduced pressure toobtain a syrup of 4-phenyl-ethyl 2-hydroxybutanoate. The syrup wasdissolved in ethanol, and an optical purity was analyzed by using highperformance liqud chromatography CHIRAL CELL OD (25 cm×0.46 cm)manufactured by DAICEL CHEMICAL INDUSTRIES LTD. Analytical conditions:eluent: hexane:isopropanol (100:1), flow rate: 0.5 ml, columntemperature: 25° C., detector: UV; 250 nm.

As a result, a reaction rate of DH5α (pKK-EnHCH1) was improved ascompared to that of the gene donor (DS-S-75 strain belonging to thegenus Enterobacter). After the reaction, measured results of an opticalpurity of the remaining R-4-phenyl-ethyl 2-hydroxybutanoate, and a yieldthereof when an amount of the racemic ethyl lactate before initiation ofthe reaction as 100% are shown in Table 8.

TABLE 8 4-phenyl-ethyl 2- hydroxybutanoate Optical purity (% ee) Yield(%) DH5α (pKK-EnHCH1) (24 hr) 98.1 (R) 9.6 (substrate 2%) DH5α(pKK-223-3) (24 hr) 0 100 (substrate 2%) DS-S-75 (48 hr) 5.27 (R) 73.3(substrate 2%) DS-S-75 (100 hr) 98.1 (R) 9.6 (substrate 2%)

By using DH5α (pKK-EnHCH1), R-methyl 4-chloro-3-hydroxybutanoate,S-3-hydroxy-γ-butyrolactone, S-ethyl 3-hydroxybutanoate,R-3-hydroxybutyric acid, R-ethyl 2-hydroxybutanoate, S-2-hydroxybutyricacid, R-methyl tetrahydrofuran-2-carboxylate, R-ethyl lactate andR-ethyl 4-phenyl-2-hydroxybutanoate can be produced. And yet, theyshowed stereoselectivity substantially equal or more than that of thegene donor (DS-S-75 strain belonging to the genus Enterobacter), and thereaction rate was extremely rapid. That is, by incorporating a genewhich encodes EnHCH, a recombinant E. coli having high stereoselectivehydrolysis activity can be obtained. In particular, hydroxycarboxylicesters as shown in Example 7 (1) to (3) had extremely highstereoselectivity.

1. An isolated gene comprising a nucleotide sequence selected from: (a)a nucleotide sequence described in SEQ. ID. NO: 1, (b) a nucleotidesequence which encodes a protein having a chlorohydrin andhydroxycarboxylic ester asymmetric hydrolase activity and hybridizeswith the nucleotide sequence described in SEQ. ID. NO: 1 under stringentconditions, wherein the stringent conditions comprise hybridizationusing a DNA derived from a colony or plaque, or a filter to which afragment of the DNA is fixed, in the presence of 0.7 to 1.0 M NaCl at65° C., and then washing the filter with 0.1 to 2-fold of a SSC solutionat 65° C., (c) a nucleotide sequence which encodes an amino acidsequence described in SEQ. ID. NO: 2, and (d) a nucleotide sequencewhich encodes an amino acid sequence having a chlorohydrin andhydroxycarboxylic ester asymmetric hydrolase activity where the aminoacid sequence is an amino acid sequence having 95% or more homology withthe amino acid sequence described in SEQ. ID. NO:
 2. 2. An isolated DNAwhich consists of the nucleotide sequence described in SEQ. ID. NO: 8.3. A vector containing the gene according to claim
 1. 4. A vectorcontaining an isolated gene comprising a nucleotide sequence selectedfrom: (a) a nucleotide sequence described in SEQ. ID. NO: 1, (b) anucleotide sequence which encodes a protein having a chlorohydrin andhydroxycarboxylic ester asymmetric hydrolase activity and hybridizeswith the nucleotide sequence described in SEQ. ID. NO: 1 under stringentconditions, wherein the stringent conditions comprise hybridizationusing a DNA derived from a colony or plaque, or a filter to which afragment of the DNA is fixed, in the presence of 0.7 to 1.0 M NaCl at65° C., and then washing the filter with 0.1 to 2-fold of an SSCsolution at 65° C., wherein said vector further contains the DNA asdefined in claim
 2. 5. The vector according to claim 4, wherein saidvector is plasmid pKK-EnHCH1.
 6. An isolated transformed cell containingthe gene as defined in claim
 1. 7. An isolated transformed cellcontaining the vector as defined in claim
 5. 8. An isolated transformedcell containing the vector as defined in claim
 4. 9. The transformantaccording to claim 6, wherein the host is E. coil.
 10. The transformantaccording to claim 7, wherein the host is E. coli.
 11. The transformantaccording to claim 8, wherein the host is E. coli.
 12. The transformantaccording to claim 6, wherein the host is E. coli JM109 strain or DH5αstrain.
 13. The transformant according to claim 7, wherein the host isE. coli JM109 strain or DH5α strain.
 14. The transformant according toclaim 8, wherein the host is E. coli JM109 strain or DH5α strain.
 15. Aprocess for the preparation of a protein comprising an amino acidsequence selected from: (a) an amino acid sequence described in SEQ. ID.NO: 2, and (b) an amino acid sequence having a chlorohydrin andhydroxycarboxylic ester asymmetric hydrolase activity where the aminoacid sequence is an amino acid sequence having 95% or more of homologywith the amino acid sequence described in SEQ. ID. NO: 2, comprisingculturing the isolated transformed cell of claim 6.